Construction Of Recombinant Strain For High Expression Of Alkaline Protease And Research On Microencapsulation

The authors acknowledge the financial support provided by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period(2012BAD33B03). The alkaline protease gene, Apr, was from Bacillus licheniformis 2709. Construction and identification of E. coli Top 10-p GM-T-Apr and E. coli BL21-p ET-28b(+)-Apr, optimization of alkaline protease producted from a recombinant strain, and alkaline protease immobilization were studied by PCR technology, Image J analysis software, SDS-PAGE technique and Encapsulator B-395 Pro. In this study, a recombinant strain to highly yield alkaline protease was successfully constructed. The protease activity, growth state of a recombinant strain and glucose utilization rate were optimized. Alkaline protease microcapsules were better than alkaline protease at stability that would contribute to their store. The results were as following:(1) Based on the protease gene sequence of Bacillus licheniformis strain 2709 announced in NCBI, the primer sequences were designed with restriction sites of Nco I and Xho I. The Apr gene was obtained by PCR with the optimized condition. Apr gene was connected into a vector p GM-T. The recombinant strain Top 10-p GM-T-Apr was successfully constructed. The Apr gene, restricted by FD.Xho I and FD.Nco I, was displayed as long as expected.(2) The alkaline protease gene, correctly identified, was connected into an expression vector p ET-28b(+), to get the recombinant plasmid p ET-28b(+)-Apr. Then the p ET-28b(+)-Apr was transferred in a strain E. coli Top 10. The sequence of the plasmid extracted from recombinant strain was proved the similarity achieved 98% as Apr from Bacillus licheniformis strain 2709. SDS-PAGE of product analysis indicated a protein of Mr of 36 k Da.(3) The glucose concentration, peptone concentration, volume of salt solution, incubation time and agitation frequency were considered in the One-Factor-at-a-Time plan. The peptone concentration, incubation time and agitation frequency were chosen to carry out response surface methodology plan. The condition to produce protease by recombinant strain with three indicators was to be obtained at: the peptone concentration 8.1 g/L, the incubation time 24.25 h and the agitation frequency 180 r/min. In this study, alkaline protease activity of recombinant strain was higher than B. Licheniformis 2709 with the same density, as the same time, the glucose utilization changed inconspicuously. Thus the recombinant strain has more widely application.(4) In order to achieve the encapsulated rate of microcapsules mathematical model, the sodium alga acid concentration, the anhydrous calcium chloride concentration, placed time and the alkaline protease concentration were studied by One-Factor-at-a-Time and response surface methodology. The experimental data were fitted to a second-order polynomial equation and profiled into the corresponding contour plots. The results showed that the optimal condition obtained by RSM is as follows: sodium alga acid concentration 1.47%, anhydrous calcium chloride concentration 2.93%, placed time 62 min and alkaline protease concentration 0.78%. Under this condition, the encapsulated rate of microcapsules achieves 95.51%. There were some differences between AP and AP microcapsules for enzymatic hydrolysis. AP microcapsules were better than AP at stability that would contribute to their store.